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Transcript
Next Generation Quantitative PCR Real-Time PCR or quantitative PCR (qPCR) is a PCR-based technique that is able to simultaneously amplify and detect changes in the amplicon concentration. Real-Time PCR collects data during PCR amplification by utilizing fluorescence signal emitted by either DNA binding dyes such as SYBR Green Dye, or special probes such as TaqMan probe. abm selection of EvaGreen qPCR MasterMixes and TaqProbe qPCR MasterMixes provides various options for both methods which also accommodates most qPCR thermal instrument models available on the market. EvaGreen qPCR MasterMixes abm’s EvaGreen 2X qPCR MasterMix provides all ingredients Reverse Transcriptase (RTase) is a well-known potent inhibitor of necessary for quantitative PCR in a premixed and optimized format. PCR. abm’s EvaGreen 2X qPCR MasterMix outperforms competi- A new generation of SYBR Green I, the EvaGreen Dye used in our tors with its greater range of compatibility to commercially available EvaGreen qPCR MasterMixes works in a similar mechanism RTases. Compared to other leading brands, our OneScript® and (Figure1). Available with either ROX or fluorescein as the internal OneScript® Plus RTases also demonstrate the least amount of inhibi- passive reference dye, abm’s EvaGreen 2X qPCR MasterMix offers tion to downstream qPCR applications (Figure 2A-E). unparalleled performance in sensitivity, signal-to-noise ratio, and complete elimination of primer dimers. • Streamlined protocol in a simple reaction set-up • Accurate quantification of a variety of gene targets abm's OneScript® abm's OneScript® Plus LifeTech's SuperScript III NEB's M-MuLV H Minus Thermo's Maxima • Reduced risk of contamination with less pipetting steps • Compatibility with most real-time PCR instruments Denaturation Polymerization Forward primer Figure 2A: With abm’s EvaGreen 2X qPCR MasterMix abm's OneScript® abm's OneScript® Plus LifeTech's SuperScript III NEB's M-MuLV H Minus Thermo's Maxima Reverse primer Polymerization Completed Figure 2B: With ABI Power SYBR Green Master Mix Figure 1: EvaGreen method: When the DNA polymerase synthesizes the complementary strand to the template, the intercalating dye, EvaGreen, incorporates itself into the dsDNA and emits a strong fluorescent signal which can be detected by the thermal cycler. abm's OneScript® abm's OneScript® Plus LifeTech's SuperScript III NEB's M-MuLV H Minus Thermo's Maxima Figure 2C: With Biorad iQ SYBR Green Supermix Next Generation Quantitative PCR abm's OneScript® abm's OneScript® Plus LifeTech's SuperScript III NEB's M-MuLV H Minus Thermo's Maxima Figure 2D: With Takara SYPR Premix Ex Taq II abm's OneScript® abm's OneScript® Plus LifeTech's SuperScript III NEB's M-MuLV H Minus Thermo's Maxima Figure 2E: With Roche FastStart SYBR Green Master Figure 2 (A-E): 20 μl RT reactions (100 pg of Human RNA per reaction) were performed with various RTases available on the market. 1 μl of each RT product was then used directly as template for GAPDH amplification using qPCR MasterMixes from various leading brands. abm’s EvaGreen 2X qPCR MasterMix is the ultimate solution for reducing non-specification amplification in qPCR. Its optimized buffer system works in tandem with a highly efficient HotStart DNA polymerase to minimize signal-to-noise ratio (Figure 3A-E) Figure 3A: abm’s EvaGreen Figure 3B: ABI Power SYBR Figure 3C: Biorad iQ Figure 3D: Takara SYPR 2X qPCR MasterMix Green MasterMix SYBR Green SuperMix Premix Ex Taq II Figure 3 (A-E): Melt curves of the corresponding MasterMixes from the reactions shown in Figure 3A-3E. Figure 3E: Roche FastStart SYBR Green Master EvaGreen Express qPCR MasterMixes abm’s EvaGreen Express 2X qPCR MasterMix provides all ingrediActivation Amp. cycle ents necessary for extremely rapid quantitative PCR in a premixed and optimized format. Our proprietary TaqFast DNA Polymerase shortens reaction time to 1/5 of standard qPCR time. Available with EvaGreen Express 2X qPCR either ROX or fluorescein as the internal passive reference dye, 95°C 30 s Melt. Curve 95°C 1-5 s 1X 40X 60°C 10 s 1X abm’s EvaGreen Express 2X qPCR MasterMix offers unparalleled sensitivity, signal-to-noise ratio, and complete elimination of primer Activation dimers. • Extremely rapid activation – only 30 seconds! • Ultra-fast qPCR thermal cycling – 10 second annealing/elongation time • Streamlined protocol with a simple reaction set-up • Ultra-fast quantification of a variety of gene targets • Reduces pipetting steps to minimize the risk of contamination • Compatible with most real-time PCR instruments. EvaGreen 2X qPCR Standard protocol 95°C 10 m Amp. cycle Melt. Curve 95°C 15 s 60°C 60 s 1X 40X 1X Next Generation Quantitative PCR TaqProbe qPCR MasterMixes One-Step EvaGreen/TaqProbe qRT-PCR Kit abm’s TaqProbe 2X qPCR MasterMix is designed for high through- Convenient real-time RNA quantification in one step. put quantitative PCR using TaqMan ® probe-based chemistry. (Figure 4) Available with either ROX or fluorescein as the internal passive reference dye, abm’s TaqProbe 2X qPCR MasterMix offers superb performance in sensitivity and signal-to-noise ratio. The multiplex formulation supports quantitative amplification and detection of up to four targets simultaneously with consistent and reliable results. • Improved fidelity and yield for reverse transcription • Prevention of template (RNA) degradation with RNaseOFF Ribonuclease Inhibitor • Superb performance with respect to sensitivity and signal-to-noise ratio • Significant reduction in non-specific PCR amplification by utilizing HotStart Taq DNA polymerase • Streamlined protocols with its single tube set-up • Fully optimized for detection of low-copy genes • Accurate quantification of a variety of gene targets - high-quality, full-length cDNA from as little as 0.01pg of RNA • Reduce contamination risks with less pipetting steps • Streamlined protocol in a simple single-tube reaction set-up • Compatible with most real-time PCR instruments • Reduced risk of contamination with less pipetting steps Forward primer R Probe Q One-Step qRT-PCR Kit Polymerization Reverse primer R Q + RNA + Gene-Specific Primer + qRT-PCR Enzyme Mix + EvaGreen / TaqProbe qPCR Mastermix 1 qRT-PCR abm G471-X G493-X One-Step qRT-PCR Strand displacement R Q Cleavage R Q Polymerization Completed Figure 4: TaqProbe method: 5’ to 3’ exonuclease activity of Taq DNA polymerase displaces the reporter dye of the TaqMan Probe, and cleaves the reporter at the 5’ end from the quencher at the 3’ end to emit fluorescence Figure 5 : 20 μl One-Step qRT-PCR reactions (20 ng, 2 ng, 200 pg, 20 pg or 2 pg of RNA per reaction) were carried out with abm’s One-Step EvaGreen qRT-PCR kit for GAPDH amplification. ExCellenCT One-Step EvaGreen/TaqProbe qRT-PCR Kit Designed for quantitative real-time analysis of miRNA samples, abm’s ExCellenCT Lysis Kit prvides an unique and effective alterna- abm’s EvaGreen miRNA qPCR MasterMix offers superb perfor- tive method to extract and process RNA templates directly from mance in sensitivity, signal-to-noise ratio, and reduction of non-spe- cultured cells in less than 15 minutes. proprietary qRT-PCR Enzyme cific PCR amplication. The proprietary formulation of our Master- Mix contains stabilizers and enhancers to optimize the two reactions Mix is optimized for the amplification of cDNA generated from in a real-time "single step". Coupled together, this complete system abm’s miRNA cDNA synthesis kits. Available with either ROX or provides the ultimate convenience in generating consistent, fluorescein as the internal passive reference dye, EvaGreen miRNA reproducible, and accurate results from 10-105 of cultured cells. qPCR MasterMixes are compatible with a range of qPCR machines. Cell Lysis Incubate for 8 min. RT from Lysates RT-PCR Analysis Add stop solution incubate 2 min. qPCR Control Template Kit abm’s qPCR Control Template Kit is a complete system including a RNA 5’ human RNA template, cDNA template and a β-actin primer mix, 3’ 5’ 3’ RT Primer allowing the user to easily incorporate a reliable positive control into abm’s ExCellenCT Lysis Kit 5X All-In-One RT MasterMix (with ExCellenCT Lysys Kit) EvaGreen miRNA qPCR MasterMixes EvaGreen 2X qPCR MasterMix and TaqProbe 2X qPCR MasterMix ExCellenCT One-Step EvaGreen qRT-PCR Kit and ExCellenCT One-Step TaqProbe qRT-PCR Kit Two-Step RT-PCR Kit their existing qPCR setups. The kit can also be used as a convenient One-Step RT-PCR Kit tool for demonstration purposes within teaching laboratories Next Generation Quantitative PCR qPCR Lentivirus Titration (Titer) Kit qPCR Adeno-Associated Virus Titration Kit Designed with titer primers for all HIV-1 based vector detection, The qPCR Adeno-Associated Virus Titration (Titer) Kit provides a abm’s Lentivirus Titration Kit enables fast and simple lentivirus fast and simple way to perform Adeno-Associated Virus titration. titration. Its rapid RNA extraction method and qRT-PCR based viral abm’s kit has demonstrated itself to be superior in both sensitivity RNA quantification allow for assay completion in just 2 hours. Along and specificity compared to similar products on the market, while with our online titer calculator, titration of lentiviral preparations has keeping non-specific background signals to a minimum. never been easier. • No RNA purification - saves time and eliminates inaccuracies • No RNA purification - saves time and eliminates inaccuracies • Ready-to-use reagent mix - reduces variability • Ready-to-use reagent mix- reduces variability • qRT-PCR in one step - more sensitive and accurate than other • Rapid set-up, qPCR assay can be run in under 2 hours methods • No NTC amplification - the only one on the market that eliminates NTC signal due to our unique primer design. abm’s qPCR Product List Cat. No MasterMix-R, LR, iC, S Product Name Size EvaGreen 2X qPCR MasterMix 500 Rxn (20 μl), 4 x 1.25 ml EvaGreen 2X qPCR MasterMix 2000 Rxn (20 μl), 16 x 1.25 ml MasterMix-5R, 5L, 5C, 5S EvaGreen 5X qPCR MasterMix 1000 Rxn (20 μl), 4 x 1.0 ml MasterMix-ER, EL, EC, ES EvaGreen Express 2X qPCR MasterMix 500 Rxn (20 μl), 4 x 1.25 ml G471-R, LR, iC, S One-Step EvaGreen qRT-PCR Kit 100 Rxn (20 μl/rxn) G917-R, LR, iC, S ExCellenCT One-Step EvaGreen qRT-PCR Kit 25 Preps, 100 x 20 μl Rxn MasterMix-P, PL, PC, PS, PM TaqProbe 2X qPCR MasterMix 500 Rxn (20 μl), 4 x 1.25 ml G493-P, PL, PC, PS One-Step TaqProbe qRT-PCR Kit 100 Rxn (20 μl/rxn) G918-P, PL, PC, PS ExCellenCT One-Step TaqProbe qRT-PCR Kit 25 Preps, 100 x 20 μl Rxn G929 qPCR Control Template Kit 100 Rxn MasterMix-mR, mL, mC, mS EvaGreen miRNA qPCR MasterMix 500 Rxn (20 μl), 4 x 1.25 ml LV900 qPCR Lentivirus Titration(Titer) Kit 100 Rxn G931 qPCR Adeno-Associated Virus Titration (Titer) Kit 100 Rxn MasterMix-R-XL, LR-XL, iC-XL, S-XL R : ROX, L : Low ROX, C : Fluorescein, S : No-dye Applied Biological Materials Inc. #1-3671 Viking Way Richmond, BC V6V 2J5 CANADA www.abmgood.com T : 02 3462 8658 www.itsbio.co.kr F : 02 3462 8659 [email protected]